Biology subjects

Puhach, O.

Publications and source records attributed to Puhach, O..

3 recordsLinked to original sources

bioRxiv2024

Molecular cloning and host range analysis of three cytomegaloviruses from Mastomys natalensis

Herpesvirus-based vectors are attractive for use as conventional or transmissible vaccines against emerging zoonoses in inaccessible animal populations. In both cases, cytomegaloviruses as members of the subfamily Betaherpesvirinae are particularly suitable for vaccine development as they are highly specific for their natural host species, infect a large proportion of their host population, and cause mild infections in healthy individuals. The Natal multimammate mouse (Mastomys natalensis) is the natural reservoir of Lassa virus, which causes deadly hemorrhagic fever in humans. M. natalensis was recently reported to harbor at least three different cytomegaloviruses (MnatCMV1, MnatCMV2 and MnatCMV3). Herein, we report the molecular cloning of three complete MnatCMV genomes in a yeast and bacterial artificial chromosome (YAC-BAC) hybrid vector. Purified viral genomes were cloned in yeast by single-step transformation-associated recombination (STAR cloning) and subsequently transferred to Escherichia coli for further genetic manipulation. Integrity of the complete cloned viral genomes was verified by sequencing, and replication fitness of viruses reconstituted from these clones was analyzed by replication kinetics in M. natalensis fibroblasts and kidney epithelial cells. We also found that neither parental nor cloned MnatCMVs replicated in mouse and rat fibroblasts, nor did they show sustained replication in baby hamster kidney cells, consistent with the expected narrow host range for these viruses. We further demonstrated that an exogenous sequence can be inserted by BAC-based mutagenesis between open reading frames M25 and m25.1 of MnatCMV2 without affecting replication fitness in vitro, identifying this site as potentially suitable for the insertion of vaccine target antigen genes. ImportanceCytomegaloviruses recently discovered in the Natal multimammate mouse (Mastomys natalensis) are widespread within the M. natalensis population. Since these rodents also serve as natural hosts of the human pathogen Lassa virus (LASV), we investigated the potential suitability of M. natalensis CMVs (MnatCMVs) as vaccine vectors. We describe the cloning of three different MnatCMV genomes as bacterial artificial chromosomes (BACs). Replicative capacity and species specificity of these BAC-derived MnatCMVs were analyzed in multiple cell types. We also identified a transgene insertion site within one of the MnatCMV genomes suitable for the incorporation of vaccine target antigens. Together, this study provides a foundation for the development of MnatCMVs as transmissible MnatCMV-based LASV vaccines to reduce LASV prevalence in hard-to-reach M. natalensis populations and thereby zoonotic transmission to humans.

microbiology↗

bioRxiv2024

Immune escape of Omicron lineages BA.1, BA.2, BA.5.1, BQ.1, XBB.1.5, EG.5.1 and JN.1.1 after vaccination, infection and hybrid immunity

In the 5th year after the emergence of SARS-CoV-2, Omicron lineages continue to evolve and cause infections. Here, we used eight authentic SARS-CoV-2 isolates to assess their capacity to escape immunity of different exposure histories and their replicative capacity in polarized human airway epithelial cells (HAE) derived from the nasal and bronchial epithelium. Using live-virus neutralization assays of 108 human sera or plasma of different immunological backgrounds, progressive immune escape was observed from B.1 (ancestral virus) to EG.5.1, but no significant difference between EG.5.1 and JN.1.1. Vaccinated individuals without natural infection and individuals with a single infection, but no vaccination showed markedly reduced or completely lost neutralization against the latest variants, while in those with hybrid immunity almost all sera showed some neutralization capacity. Furthermore, although absolute titers differed between groups, the pattern of immune escape between the variants remains comparable with strongest loss of neutralization observed for the latest variants. In vitro studies with HAE at 33{degrees}C and 37{degrees}C showed some, but minor differences in virus replication and innate immune responses upon infection. Notably, infection with XBB.1.5, EG.5.1 and JN.1.1 showed slightly increased viral growth in nasal HAE at 33{degrees}C. Altogether, these data underscore increasing immune escape across heterogeneous immunological backgrounds with gradually increasing antibody escape of evolving Omicron lineages until variant EG.5.1, but not any further for the latest dominant lineage JN.1.1. They also suggest that viral dynamics within Omicron lineages are driven by a combination of immune evasion and increase in viral replication.

microbiology↗

bioRxiv2020

Murine cytomegaloviruses m139 targets DDX3 to curtail interferon production and promote viral replication

Cytomegaloviruses (CMV) infect many different cell types and tissues in their respective hosts. Monocytes and macrophages play an important role in CMV dissemination from the site of infection to target organs. Moreover, macrophages are specialized in pathogen sensing and respond to infection by secreting cytokines and interferons. In murine cytomegalovirus (MCMV), a model for human cytomegalovirus, several genes required for efficient replication in macrophages have been identified, but their specific functions remain poorly understood. Here we show that MCMV m139, a gene of the conserved US22 gene family, encodes a protein that interacts with the DEAD box helicase DDX3, a protein involved in pathogen sensing and interferon (IFN) induction, and the E3 ubiquitin ligase UBR5. DDX3 and UBR5 also participate in the transcription, processing, and translation of a subset of cellular mRNAs. We show that m139 inhibits DDX3-mediated IFN-{beta} induction and is necessary for efficient viral replication in bone-marrow derived macrophages. In vivo, m139 is crucial for viral dissemination to local lymph nodes and to the salivary glands. An m139-deficient MCMV also replicated to lower titers in SVEC4-10 endothelial cells. This replication defect was not accompanied by increased IFN-{beta} transcription, but was rescued by knockout of either DDX3 or UBR5. Moreover, m139 co-localized with DDX3 and UBR5 in viral replication compartments in the cell nucleus. These results suggest that m139 inhibits DDX3-mediated IFN-{beta} production in macrophages and antagonizes DDX3 and UBR5-dependent functions related to RNA metabolism in endothelial cells. Author SummaryHuman cytomegalovirus is an opportunistic pathogen that causes severe infections in immunocompromised individuals. The virus infects certain types, such as macrophages and endothelial cells, to ensure its dissemination within the body. Little is known about the viral factors that promote a productive infection of these cell types. The identification of critical viral factors and the molecular pathways they target can lead to the development of novel antiviral treatment strategies. Using the mouse cytomegalovirus as a model, we studied the viral m139 gene, which is important for virus replication in macrophages and endothelial cells and for dissemination in the mouse. This gene encodes a protein that interacts with the host proteins DDX3 and UBR5. Both proteins are involved in gene expression, and the RNA helicase DDX3 also participates in mounting an innate antiviral response. By interacting with DDX3 and UBR5, m139 ensures efficient viral replication in endothelial cells. Importantly, we identify m139 as a new viral DDX3 inhibitor, which curtails the production of interferon in macrophages.

microbiology↗